Poster Presentation 38th Lorne Cancer Conference 2026

Utilising Cas gene editing systems to identify non-coding lncRNA dependencies in high-risk MLL-rearranged acute myeloid leukaemia (#158)

Steven He 1 , Richard B Lock 1 , Paul Ekert 1 , Patrick Connerty 1 , Antoine de Weck 1
  1. Childrens Cancer Institute Australia, UNSW Sydney, NSW, Australia

Background:

MLL (KMT2A) rearranged acute myeloid leukaemia (MLLr-AML) is an AML subtype with generally poor outcomes, showing only 30-54% 5-year event free survival whilst presenting in 20-25% of paediatric AML cases1-2. Chemotherapy remains the standard of care treatment but is limited by toxicity and non-specificity, underscoring the need for novel therapeutic targets3. Whilst resources such as the Cancer Dependency Map provide protein-coding dependency data in AML cell-line models4, there is currently little data concerning the non-coding genome. With recent findings demonstrating that long non-coding RNA (lncRNA) expression can independently predict survival of paediatric MLLr-AML patients5, we aimed to utilise CRISPR genome-wide screening to identify potential novel lncRNA dependencies in MLLr-AML.

Methods:

CRISPR-Cas9 and CRISPR-Cas13d expressing MLL-r AML cell lines were stably engineered via lentiviral transduction, and Cas protein activity was validated in three MLLr-AML cell lines. In vitro pooled genome-wide Cas9 screening (500X coverage) was performed over 21 days to identify dropout of single guide RNAs (sgRNAs), which was assessed using MAGeCK6 with significance cut offs of <-1.5 LFC and FDR <0.01. These hits were further validated via flow cytometry-based viability assays following induction of sgRNAs in a single-target fashion utilising Cas9, with two candidate genes taken forward and similarly investigated with a Cas13d system.  Publicly available lncRNA and healthy expression data was assessed with custom R Scripts.

Results:

Genome-wide CRISPR screening identified five novel lncRNA dependencies overlapping across all three cell lines. Single-target Cas9 KO assessed their role in AML survival over 21 days, observing a strong reduction (25-50%) in viability of cells expressing Cas9 sgRNAs targeting candidate genes. Two candidate lncRNA genes were taken forward for further study. Analysis of public datasets demonstrated elevated expression of these lncRNAs in primary AML samples compared to other cancer types, as well as compared to healthy whole blood (p<0.001), suggesting their potential as AML-specific therapeutic targets. Orthogonal Cas13d-mediated knockdown of these two candidates similarly inhibited cell growth and showed up to two-fold reduction in viability compared to controls, in agreement with our Cas9 results. These results suggest two new potential lncRNA dependencies in MLLr-AML.

 

References

  1. Van Weelderen, R. E. et al. Measurable Residual Disease and Fusion Partner Independently Predict Survival and Relapse Risk in Childhood KMT2A -Rearranged Acute Myeloid Leukemia: A Study by the International Berlin-Frankfurt-Münster Study Group. JCO 41, 2963–2974 (2023).
  1. Romy E. van Weelderen et al. Measurable Residual Disease and Fusion Partner Independently Predict Survival and Relapse Risk in Childhood KMT2A-Rearranged Acute Myeloid Leukemia: A Study by the International Berlin-Frankfurt-Münster Study Group. J Clin Oncol 41, 2963-2974(2023).
  2. Conneely, S. E. & Stevens, A. M. Acute Myeloid Leukemia in Children: Emerging Paradigms in Genetics and New Approaches to Therapy. Curr Oncol Rep 23, 16 (2021).
  3. Arafeh, R., Shibue, T., Dempster, J.M. et al. The present and future of the Cancer Dependency Map. Nat Rev Cancer 25, 59–73 (2025).
  4. Farrar, J. E. et al. Long Noncoding RNA Expression Independently Predicts Outcome in Pediatric Acute Myeloid Leukemia. JCO 41, 2949–2962 (2023).
  5. Li, W., Xu, H., Xiao, T. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol 15, 554 (2014).